Novel, esterase, fungus capable of producing the same and method for producing the same

ABSTRACT

The present invention provides a novel esterase derived from  Ideonella  sp. 0-0013 strain (FERM BP-08660) having the following properties: (1) function, substrate specificity: hydrolyzes methyl 3-hydroxypalmitate to generate 3-hydroxypalmitic acid and methanol; (2) optimal temperature for functioning: 37° C.; (3) optimal pH and stable pH range: pH 7 or more to pH 10 or less; (4) temperature stability: 97% of the enzyme is stable at 43° C.; (5) inhibition, activation, and stabilization: activated by sodium ion and potassium ion, and inhibited by strontium ion, iron ion (divalent), and methyl palmitate; (6) molecular weight: about 46,500 Da (by SDS-PAGE), about 41,000 Da (by a gel filtration method); and (7) isoelectric point: pI 4 (by polyacrylamide gel isoelectric focusing method); a microorganism producing the enzyme; and a method of producing the enzyme.

TECHNICAL FIELD

The present invention relates to a novel esterase, a microorganismproducing said enzyme and a method of producing said enzyme. This enzymehas an effect of restraining pathogenic expression of Ralstoniasolanacearum.

BACKGROUND ART

Bacterial wilt, which brings down serious damages to agricultural crops,is known to be a disease that is difficult to prevent. Methods ofpreventing bacterial wilt thus far developed include mainly a technologyaimed at killing Ralstonia solanacearum, pathogen, with antibiotics andthe like (Study Reports of Agricultural Experiment Station, OkayamaPrefectural Center for General Agriculture, No. 19, p. 29-35 (2001-12)),or a technology aimed at restraining the growth of the pathogen bygrafting using a variety that is resistant to the disease as a rootstock(Journal of Phytopathological Society of Japan, Vol. 63, No. 2, p. 83-88(1997-4)).

However, the method that involves use of antibiotics has a problem thatthe method gives the pathogen a stress to exist and a microorganismresistant to the stress will appear. On the other hand, the method thatinvolves using a variety that is resistant to the disease as a rootstocktakes many troubles and much time and hence tends to be avoided in alarge-scale cultivation.

Incidentally, it has revealed that methyl 3-hydroxypalmitate representedby the general formula (I)

functions as a regulator for the pathogenic expression of Ralstoniasolanacearum (Flavier, A. B., Clough, S. J., Schell, M. A., and Denny,T. P. (1997) Mol. Microbiol. 26, 251-259). Therefore, as Ralstoniasolanacearum invades into crops and grows therein, the concentration ofthe compound increases, thereby developing pathogenicity.

Accordingly, the above-mentioned compound is expected to be used as aprecursor for developing agricultural chemicals that are effective tobacterial wilt. The enzyme activity to decompose the compound issupposed to be applicable to a new breeding method for restraining thebacterial wilt.

However, only a chemical method such as an alkaline hydrolysis used forgeneral fatty acid esters has been available as a reaction ofdecomposing the compound, for example, a method of hydrolyzing thecompound into 3-hydroxypalmitic acid and methanol, represented by thegeneral formula (II)

Therefore, the reaction must have been performed under strong reactionconditions, including, for example, use of a strong alkali.

If an enzyme activity that catalyzes this reaction is found, hydrolysisof methyl 3-hydroxypalmitate can be carried out under mild reactionconditions unlike the chemical methods. In addition, even if other fattyacids coexist, use of an appropriate enzyme activity enables selectivehydrolysis of methyl 3-hydroxypalmitate utilizing substrate specificityof the enzyme. However, no reports on the enzyme activity to hydrolyzemethyl 3-hydroxypalmitate have been available.

Accordingly, it is an object of the present invention to provide anenzyme that hydrolyzes methyl 3-hydroxypalmitate, a microorganism thatproduces the enzyme, and a method of producing the enzyme.

The inventors of the present invention have made extensive studies witha view to achieving the above-mentioned object, and as a result, theyhave found that a microorganism isolated from soil has an ability toproduce the above-mentioned enzyme, esterase; the present invention hasbeen completed based on this finding.

DISCLOSURE OF THE INVENTION

That is, a first aspect of the present invention relates to a novelesterase, which is derived from Ideonella sp. 0-0013 strain (FERMBP-08660) and has the following properties.

-   (1) Function, substrate specificity: Hydrolyzes methyl    3-hydroxypalmitate to generate 3-hydroxypalmitic acid and methanol.-   (2) Optimal temperature for functioning: 37° C.-   (3) Optimal pH and stable pH range: pH 7 or more to pH 10 or less.-   (4) Temperature stability: 97% of the enzyme is stable at 43° C.-   (5) Inhibition, activation, and stabilization: Activated by sodium    ion and potassium ion, and inhibited by strontium ion, iron ion    (divalent), and methyl palmitate.-   (6) Molecular weight: About 46,500 Da (by SDS-PAGE), about 41,000 Da    (by a gel filtration method).-   (7) Isoelectric point: pI 4 (by polyacrylamide gel isoelectric    focusing method).

Further, a second aspect of the present invention relates to Ideonellasp. 0-0013 strain (FERM BP-08660), which has an ability to produce asubstance that hydrolyzes methyl 3-hydroxypalmitate to generate3-hydroxypalmitic acid and methanol.

Furthermore, a third aspect of the present invention relates to a methodof producing the novel esterase described above, characterized bycomprising: cultivating Ideonella sp. 0-0013 strain (FERM BP-08660) in amedium; and collecting the esterase from the culture broth.

Thus, according to the present invention, an enzyme that hydrolyzesmethyl 3-hydroxypalmitate, a microorganism that produces the enzyme, anda method of producing the enzyme are provided.

Since methyl 3-hydroxypalmitate acts as a regulator of expression of thepathogenicity in Ralstonia solanacearum, the novel esterase of thepresent invention that hydrolyzes methyl 3-hydroxypalmitate is usefulfor effectively restraining the bacterial wilt.

BEST EMBODIMENTS FOR CARRYING OUT THE INVENTION

The novel esterase of the present invention, which has theabove-mentioned properties, is a monomeric enzyme composed of a singlesubunit judging from the value of its molecular weight. This enzyme canbe obtained by cultivating Ideonella sp. 0-0013 strain in a medium. TheIdeonella sp. 0-0013 strain, a novel esterase-producing microorganism,was isolated from the soil collected in Aichi by the inventors of thepresent invention. More specifically, the strain was isolated using anisolation medium that contains methyl 3-hydroxypalmitate as a singlecarbon source.

This microorganism was identified to belong to the genus Ideonella byanalyzing the entire base sequence of 16S rDNA. The results are asfollows.

When the base sequence of 16S rDNA of 1,158 bp was determined and BLASTsearch was performed, it revealed that the strain of the presentinvention had a high homology with microorganisms belonging to the genusIdeonella, with a 99% homology with the Ideonella sp. B513 strain (astrain derived from a rice plant) and the like and with a 98% homologywith Ideonella dechloratans.

From the above-mentioned results, the Ideonella sp. strain of thepresent invention isolated from the soil as having a high activity tohydrolyze methyl 3-hydroxypalmitate was identified as the Ideonella sp.

The mycological properties of the strain of the present invention are asfollows.

The strain grows well at 30 to 43° C. on Nissui standard medium(manufactured by Nissui Pharmaceutical) to form white colonies. It alsogrows on an LB medium but slightly poorly. On an agar medium containingmethyl 3-hydroxypalmitate as a single carbon source, it grows at 43° C.It does not grow well on agar media that contain methyl palmitate ormethanol as a single carbon source, respectively.

The Ideonella sp. strain of the present invention was recognized to be anew species and deposited at International Patent Organism Depositary,National Institute of Advanced Industrial Science and Technology atTsukuba Central 6, 1-1, Higashi 1-Chome, Tsukuba-shi, Ibaraki-ken, Japanwith an Accession Number: FERM BP-08660 (transferred from FERM P-19248).In the present invention, those variants that are obtained by mutatingthe strain of the present invention naturally or with artificial meanscan be also used as far as they have an ability to produce the targetesterase.

The microorganisms that produce the novel enzyme of the presentinvention that can be used include besides the above strain of thepresent invention, genetically engineered microorganisms or plantsobtained by incorporating an enzyme gene isolated from the strain of thepresent invention into various host vector systems.

As the enzyme described in claim 1, the culture broths obtained bycultivating the above-mentioned microorganisms or the like in a mediumcan be used as they are. Alternatively, supernatants or microbial cellsrecovered from the culture broths, or their treated products can be usedas a crude enzyme.

The treated microbial cells include, for example, crushed microbialcells, cell-free extracts obtained by crushing microbial cells, andmicrobial cells treated with organic solvents such as acetone, toluene,etc. Examples of the enzyme that can be used include in addition tothose described above, a crude enzyme or a purified enzyme separatedfrom the microbial cells, immobilized microbial cells or immobilizedenzyme.

When using the enzyme, it is convenient to immobilize the enzyme to anappropriate carrier before it can be used. This facilitates separationand recovery of the enzyme from the reaction mixture after completion ofthe reaction and also reuse of the enzyme. Examples of the carrier thatcan be used include polyacrylamide and hydroxyapatite.

In the present invention, usually one kind of the crude enzyme such asmicrobial cells culture broth, microbial cells, or treated microbialcells or a purified enzyme can be used. However, two or more kindshaving similar abilities can be used in combination as desired.

Any medium can be used as the medium for cultivating the above-mentionedmicroorganisms as far as the microorganism can grow. Preferable examplesof the medium include Nissui Standard Medium (manufactured by NissuiPharmaceutical), and SCD medium (manufactured by Nihon Pharmaceutical).The microorganisms can grow well in these medium. To obtain higherenzyme activities, it is effective to add compounds having skeleton ofester of fatty acid such as methyl 3-hydroxypalmitate and methylpalmitate to the medium and utilize the compounds as an inductionsubstance for producing the enzyme.

Cultivation can be performed by a conventional method. For example,cultivation is performed at pH 4 to 10, preferably 6 to 8, at atemperature in the range of 15 to 50° C., preferably 30 to 45° C., for 6to 96 hours, preferably 12 to 30 hours aerobically. Sometimes, a higherenzyme activity can be obtained by stationary culture in a similarmanner.

After completion of the cultivation, when the enzyme is separated andcollected from the culture broth, and purified, a conventional methodmay be followed. For example, the enzyme can be purified from: asupernatant obtained by a solid-liquid separating means such ascentrifugation, from the culture broth; a cell-free extract obtained bycrushing the microbial cells; or an extract with solvent such asacetone, toluene, etc., by a conventional method such as concentration,filtration, or chromatography, etc.

The esterase of the present invention has the following properties.

-   (1) Function, substrate specificity: Hydrolyzes methyl    3-hydroxypalmitate to generate 3-hydroxypalmitic acid and methanol.

Methyl 3-hydroxypalmitate is detected by gas chromatography with acolumn of Stabilwax-DA, 30 m×0.53 mm×0.25 μm. The 3-hydroxypalmitic acidgenerated by hydrolysis of the ester can be detected by the same gaschromatography as described above.

-   (2) Optimal temperature for functioning: The optimal temperature of    the enzyme of the present invention is 37° C.-   (3) Optimal pH and stable pH range: pH 7 or more to pH 10 or less.-   (4) Temperature stability: 97% of the enzyme is stable at 43° C.,    54% of the enzyme is stable at 50° C., and 38% of the enzyme is    stable at 60° C.-   (5) Inhibition, activation, and stabilization: Activated by sodium    ion and potassium ion, and inhibited by strontium ion, iron ion    (divalent), and methyl palmitate.-   (6) Molecular weight: About 46,500 Da (by SDS-PAGE), about 41,000 Da    (by a gel filtration method).-   (7) Isoelectric point: pI 4 (by polyacrylamide gel isoelectric    focusing method).

Hereinafter, the present invention is explained in more detail by way ofexamples. However, the present invention is not limited by them.

EXAMPLE 1

200 mL of a medium containing 2.5 g/L yeast extract, 5.0 g/L peptone,and 1.0 g/L glucose was dispensed in a 500-mL Erlenmeyer flask andheated at 121° C. for 25 minutes to sterilize it. Then the Ideonella sp.0-0013 strain (FERM BP-08660) was inoculated and cultivated with shakingat 43° C. for 20 hours.

After completion of the cultivation, the culture broth was centrifugedto obtain a supernatant, which was used as a crude enzyme. For purifyingthe enzyme, 70% ammonium sulfate fractionation was performed.

TEST EXAMPLE 1

The culture broth obtained by cultivating the strain in the same manneras that in Example 1 was centrifuged to obtain a supernatant andmicrobial cells. The supernatant or the microbial cells were suspendedin 200 μL of 10 mM potassium phosphate buffer (pH 7.0) containing 0.5 mgof methyl 3-hydroxypalmitate, and the resultant was allowed to react at43° C. for 12 hours.

After completion of the reaction, 500 μL of dichloromethane was added tothe reaction mixture to extract 3-hydroxypalmitic acid. The extract wassubjected to gas chromatography (Stabilwax-DA, 30 m×0.53 mm×0.25 μm) tomeasure the decomposing activity of methyl 3-hydroxypalmitate. Theresults obtained are shown in Table 1. TABLE 1 Concentration of methylConcentration of 3-hydroxypalmitate 3-hydroxypalmitic DecompositionOperation (mmol/L) acid (mmol/L) (%) Control 6.07 Not detected —Supernatant 0.06 1.81 99 Microbial 1.43 Not detected 76 cell

TEST EXAMPLE 2

0.5 mg of methyl palmitate or methyl 3-hydroxypalmitate was reacted andanalyzed in the same manner as that in Example 1 and the decomposingactivities of the compounds were measured in each case. The resultsobtained are shown in Table 2. TABLE 2 Decomposition (%) Activity ratioMethyl 3-hydroxypalmitate 50.3 100.0 Methyl palmitate 10.3 20.5

The table shows that the hydrolytic activity for methyl3-hydroxypalmitate is about 5 times as high as that for methylpalmitate. This indicates that the enzyme activity of the presentinvention is selective to methyl 3-hydroxypalmitate.

INDUSTRIAL APPLICABILITY

According to the present invention, an enzyme that hydrolyzes methyl3-hydroxypalmitate is provided. Methyl 3-hydroxypalmitate acts as aregulator for the expression of pathogenicity in Ralstonia solanacearum.The enzyme of the present invention that hydrolyzes said compound isuseful for effectively restraining bacterial wilt.

1. A novel esterase derived from Ideonella sp. 0-0013 strain (FERMBP-08660) having the following properties: (1) Function, substratespecificity: Hydrolyzes methyl 3-hydroxypalmitate to generate3-hydroxypalmitic acid and methanol; (2) Optimal temperature forfunctioning: 37° C.; (3) Optimal pH and stable pH range: pH 7 or more topH 10 or less; (4) Temperature stability: 97% of the enzyme is stable at43° C.; (5) Inhibition, activation, and stabilization: Activated bysodium ion and potassium ion, and inhibited by strontium ion, iron ion(divalent), and methyl palmitate; (6) Molecular weight: About 46,500 Da(by SDS-PAGE), about 41,000 Da (by a gel filtration method); and (7)Isoelectric point: pI 4 (by polyacrylamide gel isoelectric focusingmethod).
 2. Ideonella sp. 0-0013 strain (FERM BP-08660) having anability to produce a substance that hydrolyzes methyl 3-hydroxypalmitateto generate 3-hydroxypalmitic acid and methanol.
 3. A method ofproducing a novel esterase of claim 1, comprising: cultivating Ideonellasp. 0-0013 strain (FERM BP-08660) in a medium; and collecting saidesterase from culture broth.